Use of giant unilamellar lipid vesicles as antioxidant carriers in in vitro culture medium of bovine embryos

Giant unilamellar vesicles (GUVs) are composed of lipophilic layers and are sensitive to the action of reactive oxygen species (ROS). The use of GUVs as microcarriers of biological macromolecules is particularly interesting since ROS produced by gametes or embryos during in vitro culture can induce the opening of pores in the membrane of these vesicles and cause the release of their content. This study investigated the behavior of GUVs [composed of 2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)] in co-culture with in vitro produced bovine embryos, as well as their embryotoxicity and effectiveness as cysteine carriers in culture medium. Embryonic developmental rates were unaffected, demonstrating the absence of toxicity of GUVs co-cultured with the embryos. No increase of intracellular ROS levels was observed in the embryos co-cultured with GUVs, indicating that the higher lipid content of the culture environment resulting from the lipid composition of the GUV membrane itself did not increase oxidative stress. Variations in the diameter and number of GUVs demonstrated their sensitivity to ROS produced by embryos cultured under conditions that generate oxidative stress. Encapsulation of cysteine in GUVs was found to be more effective in controlling the production of ROS in embryonic cells than direct dilution of this antioxidant in the medium. In conclusion, the use of GUVs in in vitro culture was found to be safe since these vesicles did not promote toxic effects nor did they increase intracellular ROS concentrations in the embryos. GUVs were sensitive to oxidative stress, which resulted in structural changes in response to the action of ROS. The possible slow release of cysteine into the culture medium by GUV rupture would therefore favor the gradual supply of cysteine, prolonging its presence in the medium. Thus, the main implication of the use of GUVs as cysteine microcarriers is the greater effectiveness in preventing the intracytoplasmic increase of ROS in in vitro produced bovine embryos.


Giant unilamellar vesicles (GUVs) are composed of lipophilic layers and are sensitive to the action of reactive oxygen species (ROS). The use of GUVs as microcarriers of biological macromolecules is particularly interesting since ROS produced by gametes or embryos during in vitro culture can induce the opening of pores in the membrane of these vesicles and cause the release of their content. This study investigated the behavior of GUVs [composed of 2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)] in co-culture
with in vitro produced bovine embryos, as well as their embryotoxicity and effectiveness as cysteine carriers in culture medium. Embryonic developmental rates were unaffected, demonstrating the absence of toxicity of GUVs co-cultured with the embryos. No increase of intracellular ROS levels was observed in the embryos co-cultured with GUVs, indicating that the higher lipid content of the culture environment resulting from the lipid composition of the GUV membrane itself did not increase oxidative stress. Variations in the diameter and number of GUVs demonstrated their sensitivity to ROS produced by embryos cultured under conditions that generate oxidative stress. Encapsulation of cysteine in GUVs was found to be more effective in controlling the production of ROS in embryonic cells than direct dilution of this antioxidant in the medium. In conclusion, the use of GUVs in in vitro culture was found to be safe since these vesicles did not promote toxic effects nor did they increase intracellular ROS concentrations in the embryos. GUVs were sensitive to oxidative stress, which resulted in structural changes in response to the action of ROS. The possible slow release of cysteine into the culture medium by GUV rupture would therefore favor the gradual supply of cysteine, prolonging its presence in the medium. Thus, the main implication of the use of GUVs as cysteine microcarriers is the greater effectiveness in preventing the intracytoplasmic increase of ROS in in vitro produced bovine embryos.
In vitro production (IVP) of bovine embryos has grown significantly in recent decades. This biotechnology has become an important tool for accelerating the genetic improvement of cattle herds and has been applied on a large commercial scale 1,2 .
The success of IVP is directly related to gamete quality 3 . However, the results are also influenced by extrinsic factors resulting from the excessive manipulation and exposure of gametes and embryos to the adverse conditions of the culture environment. The high concentrations of oxygen and the prolonged light exposure of these www.nature.com/scientificreports/   www.nature.com/scientificreports/ tive stress in cultured embryos exposed to menadione. Next, we evaluated (1) the embryotoxicity of GUVs; (2) intracytoplasmic concentrations of ROS in embryos co-cultured with GUVs, and (3) the morphometry and quantity of GUVs co-cultured with embryos subjected to menadione-induced oxidative stress. The effects of supplementation with menadione during embryo culture on embryo outputs and generation of oxidative stress are given in Fig. 3. No difference (P > 0.05) in the cleavage rate was observed between treatments, but blastocyst rate was decreased in the presence of menadione (control group: 37.24% ± 5.4 vs. MD 5.0 μM group: 15.75% ± 0.9; P < 0.05, Fig. 3a). Higher intracellular ROS concentrations (Fig. 3b) were observed in embryos of the MD 5.0 μM group (26.61 ± 3.29 AUF) when compared to the control (13.56 ± 0.99 AUF; P < 0.05).
Higher intracellular ROS concentrations (Table 1) were observed in embryos of the GUV + MD 5.0 μM group (16.28 ± 4.61 AUF), which differed (P < 0.05) from the control (3.76 ± 0.46 AUF) and GUV (3.99 ± 0.33 AUF) groups. Embryos of the GUV + MD 7.5 μM group were not evaluated because of the lack of development of an adequate number of structures for this assessment.
There was no difference in the diameter of GUVs between the different treatments at the beginning of culture (D1; P > 0.05) ( Table 2). However, an increase (P < 0.05) in GUV diameter was observed at the end of culture Data are expressed as the mean ± standard error of four independent replicates. Embryos were cultured for 7 days, and menadione (5.0 μM) was included in the medium only on the sixth day of culture (from D6 to D7). Table 1. Development and intracellular concentrations of reactive oxygen species (ROS) in bovine embryos cultured in vitro in the presence of giant unilamellar vesicles (GUVs) and subjected to menadione (MD)induced oxidative stress. Data are expressed as the mean ± standard error of four independent replicates. *Embryos were cultured for 7 days (D7), and menadione (5.0 μM or 7.5 μM) was included in the medium only on the sixth day of culture (from D6 to D7). ROS concentrations are expressed as arbitrary units of fluorescence (AUF). ab Different letters in each column indicate significant differences (P < 0.05).
No difference between treatments (P > 0.05) was found for the variation in the number of GUVs (%) present in the wells at the end of culture (D7) compared to D1, with 13.6% ± 4.9 of remnant GUVs on D7 in the GUV group, 21.6% ± 4.7 in the GUV + MD 5.0 μM group, and 13.5% ± 5.3 in the GUV + MD 7.5 μM group (Fig. 4). However, the number of remnant GUVs in the wells was decreased overtime (D1 vs. D7) in all groups (P < 0.05).

Supplementation of in vitro culture medium with antioxidant encapsulated in GUVs provides
greater protection against embryonic oxidative stress than supplementation through direct dilution in the medium. The suitability of GUVs as antioxidant carriers in embryo culture medium was evaluated in this experiment. For this purpose, the effect of the antioxidant cysteine diluted in culture medium [concentration of 0.6 mM as recommended in previous studies 14,15 was compared to that of cysteine (Cyst) encapsulated in GUVs [GUV(Cyst 3 mM)]. These GUVs were obtained by electroformation in a solution containing 3 mM cysteine (5X solution) and subsequent dilution in the culture medium (20%, v/v) in order to obtain a maximum final concentration of cysteine in the medium of 0.6 mM after complete content release from the GUVs.
Intracellular concentrations of ROS were evaluated in embryos on D7 (Table 4), except for the Cyst + MD and GUV(Cyst) + MD groups because of the lack of development of an adequate number of structures for this assessment. Embryos of the GUV(Cyst 3 mM) group had lower (P < 0.05) ROS concentrations (0.09 ± 0.01 AUF) than those of the Cyst group (0.16 ± 0.02 AUF), but neither group differed from the control group (0.10 ± 0.01 Table 2. Effect of menadione (MD) on the diameter of giant unilamellar vesicles (GUVs) co-cultured in vitro with bovine embryos. Data are expressed as the mean ± standard error of four independent replicates. # D1 = day 1 (beginning of cultivation); D7 = day 7 (end of 168-h cultivation). ## Embryos were cultured for 7 days, and menadione (5.0 μM or 7.5 μM) was included in the medium only on the sixth day of culture (from D6 to D7). ab Different letters in each column indicate significant differences (P < 0.05). The asterisk (in the same row) indicates a significant difference within the same treatment when evaluated on different days (P < 0.05).

Discussion
This is the first study that proposes the use of GUVs as antioxidant carriers for supplementation of in vitro culture media of bovine embryos. Overall, the results showed that GUVs are not embryotoxic and are sensitive to ROS produced by embryos cultured under conditions that generate oxidative stress. Additionally, encapsulation of the antioxidant in GUVs was found to be more effective in controlling the production of ROS in embryonic cells than direct dilution of the antioxidant in the medium. The present results revealed no changes in the diameter of GUVs cultured in vitro in the presence of menadione. This compound has been used in previous studies to induce oxidative stress in in vitro cultured cells 39 . It acts by disrupting the cellular pyridine nucleotide redox balance, compromising cellular ATP production and mitochondrial respiratory activity 40 . As a consequence, cells produce the main ROS such as O 2 − and H 2 O 2 41 . Although we did not investigate the presence of ROS in the culture medium, the results suggest that menadione did not induce the generation of ROS because of the absence of cells; thus, this drug did not directly trigger oxidative stress in GUVs. In a second step, we evaluated the direct effect of a free radical (H 2 O 2 ) on GUVs. This radical promoted changes in the diameter of these structures even when cultured in vitro in the absence of cells. This variation in surface area and the concomitant morphological changes in GUVs have been previously observed in response to irradiation in the presence of photooxidative substances 36 .
After determination of the sensitivity of GUVs to H 2 O 2 , these structures were co-cultured with embryos under conditions that generate oxidative stress (menadione challenge) in order to evaluate the susceptibility of GUVs to ROS produced by cultured cells. Variations in the diameter and number of remnant GUVs were observed at Table 3. Cleavage and development of bovine embryos cultured in vitro in medium supplemented with giant unilamellar vesicles (GUVs) and cysteine (Cyst) and subjected to menadione (MD)-induced oxidative stress. Data are expressed as the mean ± standard error of four independent replicates. A Embryos were cultured for 7 days, and menadione (5.0 μM) was included in the medium only on the sixth day of culture (from D6 to D7). B In the Cyst and Cyst + MD groups, cysteine was diluted directly into the culture medium at the final concentration of 0.6 mM. C In the GUV(Cyst 3 mM) and GUV(Cyst 3 mM) + MD groups, the GUVs were electroformed in a solution containing 3 mM cysteine (5X solution) and subsequently diluted in the culture medium (20% v/v) in order to obtain a maximum final concentration of cysteine in the medium of 0.6 mM after complete content release from the GUVs. ab Different letters in each column indicate significant differences (P < 0.05).  www.nature.com/scientificreports/ the end of the culture period in the group treated with the highest menadione concentration tested (7.5 µM). This result corroborates the sensitivity of GUVs to H 2 O 2 and might be explained by the fact that these vesicles, which are composed of unsaturated phospholipids, are extremely sensitive to lipid peroxidation, a chain reaction mediated by oxidative products 42 . Recent studies have shown that the exposure of GUVs to photooxidative substances leads to the cleavage of lipid chains and the consequent opening of pores in the membrane, causing changes in its structure 33,43,44 such as surface area variation, associated or not with membrane fluctuations and the release of "buds" due to oxidative stress 36,37,42 . Indeed, it has previously been reported that ROS can react with the unsaturations of the lipid chains forming hydroperoxides. The hydrophilic nature of these groups favors migration towards the polar heads of the lipids leading to an increase of the mean molecular area per lipid and the overall surface area expansion of the GUVs 33, 45 . In addition, the propagation of the oxidation reactions may lead to the chain cleavage at the unsaturation sites, inducing the pore opening in the lipid membrane 33,46 and further decrease of the GUV dimension. Although small, the reduction in population of GUVs over time is indeed significant. This effect is inherent to the stability of the colloidal system formed by self-assembled lipid structures 47 , which can be attributed to a variety of environmental conditions, such as temperature and ionic strength. Thus, our results thus suggest that the variation in the diameter of GUVs may be the result of their sensitivity to oxidative stress. Although we did not evaluate the ultrastructure of GUVs, literature data indicate that the variation in GUV diameter may be a consequence of membrane damage caused by ROS, especially by the H 2 O 2 radical 33, 35-38, 43, 44 . One may thus assume that this membrane damage promotes possible extravasation of the content of GUVs. Since GUVs are considered useful tools for macromolecule encapsulation 29 and free radicals can damage their membrane 37 , causing possible extravasation of their content, these structures are excellent candidate vehicles for the gradual release of antioxidants into the in vitro culture medium of embryos in situations that generate oxidative stress. Within this context, the supply of antioxidant to the medium via GUVs would permit a response to the specific needs of the culture system since the unnecessary or excessive inclusion of antioxidants can be harmful 19 . For example, at low concentrations, ROS play a physiological role in cells; more specifically, they regulate cell function by controlling the production and activation of substances with biological activity in the main signaling pathways 48,49 . To our knowledge, there are no studies that have co-cultured GUVs with embryos. Thus, their embryotoxicity needs to be evaluated. The present results demonstrated that GUVs did not affect embryonic development and do therefore not exert any morphologically evident embryotoxic effect. Likewise, no increase of intracellular ROS levels was observed in embryos cultured in vitro in the presence of GUVs, indicating that the higher lipid content of the culture environment resulting from the lipid composition of the GUV membrane itself did not cause any increase in oxidative stress. Based on these satisfactory results, cysteine was encapsulated in GUVs by electroformation since the presence of cysteine in the culture medium is essential for the synthesis of GSH by the embryo 14 , favoring the quality of the quality of the embryos produced. However, supplementation of the culture medium with cysteine, either diluted or encapsulated in GUVs, did not promote an increase in blastocyst production rates when the cultures were not challenged with menadione, www.nature.com/scientificreports/ although an interesting numerical increase in embryo production was observed [25.58% in the control group vs. 31.77% and 30.16% in the Cyst and GUV(Cyst 3 mM) groups, respectively]. Similarly, in the absence of oxidative stress, neither cysteine supplementation system reduced intracytoplasmic ROS levels in the embryos compared to control. According to the literature, the addition of antioxidants during in vitro culture produces contradictory results in terms of embryo development, with studies reporting beneficial 14 , undesirable 50 , or no effects 51 . It is most likely that beneficial effects are only seen when the culture conditions are inadequate. 52 Thus, the lack of effects of the antioxidant suggests that the culture conditions in this study were satisfactory and did not generate excessive oxidative stress. 48,49 In the present study, although supplementation of the medium with 0.6 mM cysteine did not compromise embryo developmental rates, the results showed that supplementation with this antioxidant encapsulated in GUVs was more effective than its direct dilution in the medium in preventing the increase of intracytoplasmic ROS levels in embryos caused by the inherent conditions of the culture system itself, i.e., in the absence of menadione [0.09 AUF in the GUV (Cyst 3 mM) vs. 0.16 AUF in the Cyst group]. One may speculate that the direct dilution of cysteine in the culture medium may have resulted in its rapid oxidation into cystine 53,54 , consequently reducing the availability of the former as precursors of GSH synthesis 17,53 . On the other hand, the possible slow release of cysteine into the culture medium by rupture of the GUVs would favor the gradual supply of cysteine, prolonging its presence in the medium. These results are promising and encourage the continuation of this study in order to implement this tool in reproductive biotechnology protocols. 17,53 In summary, we demonstrated that in vitro cultured GUVs had no toxic effect and did not increase intracellular ROS concentrations in the embryos. The use of these structures was safe for the IVP purposes described in this study. Since they are sensitive to oxidative stress, GUVs underwent structural changes in response to the action of ROS, indicating that these structures are suitable microcarriers for the on-demand release of antioxidants. Compared to the system in which cysteine was diluted directly in the medium (without encapsulation), GUVs containing cysteine were more effective in preventing the intracytoplasmic increase of ROS in in vitro produced bovine embryos. These results indicate that the use of GUVs as microcarriers is safe and beneficial for improving the quality of in vitro produced embryos. The findings should encourage new studies implementing the use of this support tool in order to improve the efficiency of reproductive biotechnologies. The in vitro maturation (IVM) medium consisted of TCM199 supplemented with 10% (v/v) fetal calf serum (FCS; Gibco BRL, Grand Island, NY, USA), 0.2 mM sodium pyruvate, 25 mM sodium bicarbonate, 50 μg/mL amikacin, 0.5 μg/mL FSH (Folltropin-V; Bioniche Animal Health, Ontario, Canada), and 100 IU/mL hCG (Vetecor; Hertape Calier, Juatuba, MG, Brazil). The fertilization medium (IVF-TALP) consisted of Tyrode's albumin lactate pyruvate (TALP) containing 0.2 mM Na-pyruvate, 6 mg/mL fraction V fatty acid-free BSA, 25 mM sodium bicarbonate, 13 mM Na-lactate, 50 μg/mL amikacin, 10 μM hypotaurine, 20 μM penicillamine, 2 μM epinephrine, and 10 μg/mL heparin. The embryo culture medium contained modified synthetic oviductal fluid (mSOF) supplemented with 0.2 mM L-glutamine, 0.34 mM sodium citrate, 2.8 mM myo-inositol, 2% essential amino acids, 1% non-essential amino acids, 0.2 mM pyruvate, 50 μg/mL amikacin, 5 mg/mL BSA, and 2.5% (v/v) FCS.

Methods
Cysteine (Sigma C7352) was freshly diluted in mSOF daily during the experiment to obtain a final concentration of 0.6 mM 14, 15 . Electroformation and evaluation of giant unilamellar vesicles. The GUVs were prepared by electroformation 33 , with some modifications 55 . Phospholipids 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Liss Rod PE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). DOPC GUVs were diluted in chloroform at a concentration of 10 −3 mol/L. Fluorescent GUVs were obtained by adding 0.5 mol% of Liss Rod PE. Next, 10 μL of the chloroform solution containing the lipid mixture was spread on the surfaces of two conductive Indium tin oxide-coated slides, which were then assembled to form a growing chamber separated by a 2-mm-thick Teflon frame. After drying under vacuum, the chamber was filled with 260 mOsm kg −1 sucrose solution that contained 3 × 10 −3 mol/L cysteine [GUV (Cyst 3 mM)] or not [GUV]. The osmolarity of the sucrose solution was set to match the osmolarity of the mSOF medium (260 mOsm kg −1 ) measured with an osmometer (Osmomat 3000). The chamber was then connected to an alternating power generator (Gerador de Função Digital Minipa MFG-4202, Minipa do Brasil Ltda) operating at 1 V and frequency of 10 Hz for 3 h.
The GUVs were evaluated under an inverted fluorescence microscope (Nikon Eclipse Ti-S) equipped with a 40× objective using excitation (515 to 575 nm) and emission (50 to 80 nm) filters. The images obtained with the Nikon NIS-Elements AR Software (version 3.0) were subsequently evaluated using the Image J program.
Oocyte collection and maturation. Cumulus-oocyte complexes (COCs) were obtained from cattle ovaries collected at a local abattoir by aspirating antral follicles (3-8 mm  In vitro fertilization and embryo culture. Following IVM, oocytes were cultured for 18 h in 500 μL IVF-TALP containing spermatozoa. Motile sperm were obtained by centrifuging frozen-thawed semen through a discontinuous Percoll (GE Healthcare, Uppsala, Sweden) density gradient (45% over 90%) at 2,500 g for 5 min at room temperature. Sperm were diluted in IVF-TALP to achieve a final concentration of 1 × 10 6 /mL in the fertilization well. After removal of cumulus cells, the presumptive zygotes were transferred to 500 μL mSOF medium (25-30 zygotes per well) in a 4-well plate, without mineral oil covering. Embryos were cultured at 38.5 °C in a humidified atmosphere of 5% CO 2 in air for up to 7 days. The day of in vitro fertilization (IVF) was defined as day 0 (D0). The cleavage rate was assessed at 72 h post-insemination (D3) and the blastocyst rate at 168 h postinsemination (D7). The D7 blastocyst rate was determined based on the number of inseminated oocytes.
Oxidative stress detection. Intracellular

Experiment 3: induction of oxidative stress by menadione in embryos cultured in vitro in the presence of GUVs.
In the first series of this experiment, presumptive zygotes produced in vitro were removed from the fertilization droplets on D1, washed, and transferred in groups of 25-30 zygotes per well to 4-well plates in 500 μL mSOF without (control, n = 219) or with 5.0 μM menadione supplementation from D6 to D7 (MD 5.0 μM, n = 172). The embryos were cultured for 7 days and the expanded D7 blastocysts were stained for oxidative stress detection. In another experiment, the presumptive zygotes were removed from the fertilization droplets on D1, washed, and transferred in groups of 25-30 zygotes per well to 4-well plates in 500 μL mSOF according to the following treatments: (1) control (n = 100): embryos cultured in mSOF; (2) GUV (n = 104): embryos cultured in mSOF plus 20% (v/v) GUVs; (3) GUV + MD 5.0 μM (n = 104): embryos cultured as described for the GUV group and supplemented with 5.0 μM menadione from D6 to D7, and 4) GUV + MD 7.5 μM (n = 100): embryos cultured as described for the GUV group and supplemented with 7.5 μM menadione from D6 to D7 39 . The embryos were cultured for 7 days without medium changes (feeding) during culture since one of the objectives of this experiment was to evaluate the morphometry and proportion of remnant GUVs at the end of culture. The diameter and quantity (expressed as percentage) of GUVs were evaluated at the beginning (D1) and end of culture (D7). The percentage of GUVs on D7 was determined in relation to the total number of GUVs present on D1. Blastocysts obtained on D7 were evaluated regarding intracellular ROS levels. The culture system was defined as embryotoxic when there was a significant decrease in the blastocyst rate compared to control. www.nature.com/scientificreports/ (from D1 to D7); (6) GUV(Cyst 3 mM) + MD (n = 113): culture in mSOF plus 20% (v/v) GUVs electroformed with 3 mM cysteine (from D1 to D7) and supplemented with 5.0 μM menadione from D6 to D7. The embryos were cultured at 38.5 °C in a 5% CO 2 atmosphere in air at maximum humidity for up to 7 days. Since one of the objectives of this experiment was to evaluate the morphometry and proportion of remnant GUVs at the end of culture, no medium changes (feeding) were performed during the culture period. Blastocysts obtained on D7 were evaluated regarding intracellular ROS levels.
Statistical analysis. The experiment was repeated at least three times for each proposed evaluation. The data were analyzed by ANOVA using the JMP 5.0.1a software (SAS Institute, Inc., Cary, NC, USA). Percentage data were arcsine transformed before being submitted to ANOVA when necessary. If a statistically significant effect was found, means were compared by Tukey's multiple comparisons test. Differences with probabilities (P) less than 0.05 were considered to be significant. Values are reported as the mean ± standard error of the mean (SEM).